Duplication of the genome by DNA replication is a prerequisite for normal cell division required for growth and development. Synthesis of DNA is catalyzed by DNA polymerases, however, these enzymes alone cannot make DNA efficiently enough to duplicate the entire genome. DNA polymerase processivity factors, a sliding clamp and clamp loader, enhance the efficiency of DNA replication by tethering a DNA polymerase to the template being copied. The structure and function of these processivity factors are conserved from bacteria to man. The clamp loader is a molecular machine that uses ATP to catalyze the assembly of ring-shaped sliding clamps onto DNA. The major goal of this proposal is to elucidate the mechanism by which the eukaryotic clamp loader, replication factor C (RFC), assembles clamps on DNA by defining functions for individual components. Our overriding hypothesis is that each interaction RFC makes with its binding partners, including individual ATP molecules, the clamp (PCNA), and DNA, induces conformational changes that facilitate the next step in the pathway, and these discrete conformational changes favor an ordered sequence of events to promote efficient clamp loading. Our major approach to testing this hypothesis will be to analyze reactions catalyzed by purified Saccharomyces cerevisiae RFC and an alternative Rad24-RFC clamp loader in vitro using fluorescence-based assays to measure proteinprotein and proteinDNA interactions as well as ATP hydrolysis. In addition, site-directed mutagenesis to conserved sequence motifs in ATP binding sites will be used to evaluate the contributions of individual RFC subunits to clamp loading. Specifically, our aims are 1) to define functions for ATP binding and hydrolysis by individual RFC subunits, 2) to use the alternative clamp loader, Rad24-RFC, as a tool to identify contributions that the large "A-subunit" of RFC makes to PCNA and DNA binding, 3) to identify reciprocal effects of clamp and DNA binding on the activities of RFC and Rad24- RFC. A major strength of our fluorescence approach is that this dynamic clamp loading reaction can be monitored directly in solution and in real time to uncover the temporal order of events, and factors that give rise to this order. Our broad and long-term objectives are to define molecular mechanisms by which the replication machinery duplicates genomes, and to define mechanisms by which these enzymes respond to DNA damage that is encountered during replication. This project will contribute to those objectives by characterizing the biochemical activities of DNA polymerase processivity factors, RFC and PCNA, and of a DNA damage checkpoint complex, Rad24-RFC. A fundamental understanding of the biochemical basis of DNA replication is essential to making clinical correlations between biochemical defects and disease. Basic research in the area of DNA replication has led to the development of important medical diagnostic tools as well as the development of therapeutic agents that inhibit replication of pathogens. [unreadable] [unreadable] [unreadable]